Protracted GLP-1 compositions

ABSTRACT

Glucagon-like peptide-1 (GLP-1) compositions having protracted action and methods for treating diabetes mellitus with same. Thixotropic GLP-1 compositions may further contain a phenolic or alcoholic aromatic compound, a preservative, and or divalent metal compounds.

FIELD OF THIS INVENTION

[0001] The present invention relates to a composition containing GLP-1compounds having protracted action and to a process for preparationthereof.

BACKGROUND OF THIS INVENTION

[0002] Diabetes is characterized by an impaired glucose metabolismmanifesting itself among other things by an elevated blood glucose levelin the diabetic patients. Underlying defects lead to a classification ofdiabetes into two major groups, i.e. type I and type II diabetes. Type Idiabetes, also designated insulin demanding diabetes mellitus (IDDM),arises when patients lack β-cells producing insulin in their pancreaticglands. Type II diabetes, also designated non-insulin dependent diabetesmellitus (NIDDM), occurs in patients with an impaired β-cell functionbesides a range of other abnormalities.

[0003] Type I diabetic patients are currently treated with insulin whilethe majority of type II diabetic patients are treated either with agentsthat stimulate β-cell function or with agents that enhance the tissuesensitivity of the patients towards insulin.

[0004] Glucagon-like peptide-1, also designated GLP-1, is a peptidesequence found as a constituent of mammalian proglucagon. In 1985, itwas demonstrated that GLP-1(1-36) amide stimulates insulin release fromisolated precultured rat pancreatic islets in the presence of glucose ina dose-dependent manner. This finding suggests that GLP-1(1-36) amideand related peptides might be useful in the treatment of type IIdiabetes. In recent years, particular interest has focused on GLP-1fragments such as GLP-1(7-37) and GLP-1(7-36) amide and analogues andfunctional derivatives thereof. Hereinafter, these compounds aredesignated GLP-1 compounds.

[0005] It has been found that GLP-1 compounds such as GLP-1(7-37) andGLP-1(7-36) amide have a too fast action when administered to humansubjects. Therefore, there is a need for compositions containing GLP-1compounds and having a protracted action. The availability of suchprotracted compositions will spare the diabetics the chore anddiscomfort of multiple daily injections.

[0006] Apparently, some theoretical possibilities of controlling theduration of action of GLP-1(7-37) is described at the bottom of Column 6in U.S. Pat. No. 5,120,712. The possibilities mentioned therein are theuse of polymers to complex or adsorb GLP-1(7-37), the selection ofappropriate macromolecules (for example, protamine sulphate is mentionedamong other), the incorporation of GLP-1(7-37) into particles of apolymeric material or the entrapment of GLP-1(7-37) in microcapsules.

[0007] A huge number of possible ways of preparing prolonged delivery ofcertain GLP-1 compounds is desribed in a European patent applicationhaving publication number 619,322. The possibilites mentioned thereinare to add a polymer, to prepare an oil suspension, to add zinc (II), toadd a metal, to add a basic polypeptide, to add a phenolic compound, toprepare an amorphous/crystalline formulation, or to use a liposomedelivery system.

[0008] None of these known compositions are gels or thixotropiccompositions.

[0009] One object of this invention is to provide compositionscontaining GLP-1 compounds and having a protracted action.

[0010] A further object of this invention is to provide compositionscontaining GLP-1 compounds and having a sufficient high stability, e.g.chemical stability and, especially, physical stability.

BRIEF DESCRIPTION OF THIS INVENTION

[0011] Surprisingly, it has been found that compositions containing aGLP-1 compound and a phenolic and/or an alcoholic aromatic compound incertain concentrations result in a thixotropic gel showing a protractedrelease of the active GLP-1 compound.

DETAILED DESCRIPTION OF THIS INVENTION

[0012] This invention deals with compounds having GLP-1 like activityherein referred to as GLP-1 compounds. GLP-1 compounds bind to the GLP-1receptor (vide Proc.Nat. Acad.Sci.USA 89 (1992), 8641). Examples ofspecific GLP-1 compounds are polypeptides comprising the 7-34 amino acidsequence of GLP-1, viz. formula I:

[0013]His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys(I)

[0014] or a peptide sequence derived from formula I without eliminatingthe GLP-1 like activity. The term GLP-1 compound also comprisesderivatives of said polypeptides such as acid addition salts,carboxylate salts, lower alkyl esters, amides, lower alkyl amides andlower dialkyl amides.

[0015] The compositions of this invention are gels. In a preferredembodiment, the gels have thixotropic properties. One way of preparingthe thixotropic gels according to this invention is to mix the GLP-1compound with a phenolic or an alcoholic aromatic compound in an aqueousmedium. Preferably, the phenolic or alcoholic aromatic compound is apharmaceutically acceptable antimicrobial preservative. Non-limitingexamples of such compounds include benzyl alcohol, a cresol, e.g.,m-cresol, a phenol, e.g., phenol or resorcinol, or a paraben, e.g.,methyl paraben or propyl paraben. The compositions of this invention maycontain both a phenolic or an alcoholic aromatic compound and a divalentmetal ion, preferably in the form of a salt. A preferred ion is Zn(II).Other metal ions may also be used including Ca(II), Mg(II), Co(II),Mn(II), Fe(II), and Cu(II). For example, the divalent metal salts can bethe chloride or another pharmaceutically acceptable salt. Depending onwhich process has been used during the purification of the GLP-1compound, it is, in some cases preferred to add an acetate and, in othercases, preferred to avoid the presence of acetate in the final GLP-1composition.

[0016] The compositions of this invention can be prepared by using theGLP-1 compound in a concentration within a certain range. Consequently,a preferred embodiment of this invention is compositions containing notless than about 2 mg/ml, preferably not less than about 5 mg/ml, morepreferred not less than about 10 mg/ml of a GLP-1 compound and,preferably, containing not more than about 100 mg/ml of a GLP-1compound.

[0017] Another preferred embodiment of this invention relates to athixotropic composition containing no compounds which are known to formthixotropic mixtures. It is novel that GLP-1 compounds and phenolic oralcoholic aromatic compounds which can safely be administered to humanbeings in medicaments can form thixotropic gels.

[0018] In addition to the specific ingredients which are to be presentin the compositions of this invention, said composition may also, inaddition to water, contain a pH buffering agent, an osmotic pressurecontrolling agent or other ancillary agents.

[0019] The compositions of this invention can be used as aninsulinotropic agent in the treatment of diabetes. The dosage to beadministered to human subjects is conveniently determined by aphysician. The dosage may be in the range 1-1,000 μg/kg/day. Normally,the compositions of this invention are administered subcutaneously orintramuscularly.

[0020] The features disclosed in the present description, examples andclaims may, both separately and in any combination thereof, be materialfor realizing this invention in diverse forms thereof.

[0021] This invention is further illustrated by the following exampleswhich are not to be construed as limiting, but merely as an illustrationof some preferred features of this invention. Additional preferredembodiments of this invention are stated in the claims.

[0022] Study of gelling and thixotropic properties

[0023] When evaluating compositions for their gelling and thixotropicproperties, the following tests, which are performed at a temperature of20-25° C., can be used:

[0024] Step 1: A cylindrical glass vial having a flat bottom, an innerdiameter of about 6.4 mm and a height of about 4 cm is filled to aheight of about 1 cm from the bottom with the composition which is to betested. The filling can be made using a syringe. The glass vial used canbe a 1 ml Clear Glass Vial With Caps from Waters, USA (part No. 78514).

[0025] Step 2: The vial is equipped with a cap and is stored for 24hours.

[0026] Step 3: After removal of the cap, a glass ball is very cautiouslyplaced at the top of the composition to be tested. This glass ball has aweight of about 17-18 mg and a diameter of about 2.4 mm. After standingfor 1 hour, the glass ball should not sink more than about 5 mm.

[0027] Step 4: Thereafter, the vial is placed in a vortex mixer (forexample, a whirlimixer from Fisons Scientific Apparatus, England) andshaken. During this mixing step, the ball shall drop to the bottom.

[0028] For preferred compositions according to this invention the glassball should not sink more than about 5 mm after standing for 5 hours instep 3 above, and more preferred it does not sink more than about 5 mmafter standing for 24 hours in step 3 above.

[0029] A still further feature of preferred compositions according tothis invention is that Step 4 is followed by the following steps:

[0030] Step 5: The vial is equipped with a cap and is stored for 24hours.

[0031] Step 6: Thereafter, the vial is turned upside-down.

[0032] Step 7: After standing for 1 hour, the glass ball should not sinkmore than about 5 mm.

[0033] For preferred compositions according to this invention the glassball should not sink more than about 5 mm after standing for 5 hours instep 7 above, and more preferred it does not sink more than about 5 mmafter standing for 24 hours in step 7 above.

[0034] Absorption studies.

[0035] The absorption of the GLP-1(7-37) compositions, described in theexamples, were studied in pigs after subcutaneous injection. Thecompositions were made from a mixture of GLP-1(7-37) and a trace amountof ¹²⁵I-GLP-1(7-37). One composition was injected at one side of theneck and another composition at the other side in each of 6 pigs. Theabsorption was followed by external monitoring of the radio-activityremaining at the site of injection. The injections were performed byNovo-Pen® to a depth of 5 mm.

EXAMPLE 1

[0036] The zinc free gel composition of this invention designated A was:20 mg/ml GLP-1(7-37), 16 mg/ml glycerol, and 3 mg/ml m-cresol (pH value:7.2).

[0037] This composition was made by mixing 2.5 ml acidic GLP-1(7-37)solution (20 mg/ml) with 7.5 μl of m-cresol and 40 mg of glycerol,followed by adjustment of the pH value, which was made possible by thethixotropic properties of the gel that assumed low viscosity bystirring. A high viscosity gel was formed soon after stirring wasstopped. 60 μl was injected in each pig.

EXAMPLE 2

[0038] The zinc containing gel composition of this invention designatedB was: 20 mg/ml GLP-1(7-37), 0.5 mmol/a Zn⁺⁺, 16 mg/ml glycerol, and 3mg/ml m-cresol. The molar ratio between Zn⁺⁺and GLP-1(7-37) was 0.08.

[0039] This composition was made by mixing 1 ml of GLP-1(7-37) solution(40 mg/ml), adjusted to a pH value of 7.4, with 1 ml of a solutioncontaining 6 g/l m-cresol, 32 g/l glycerol and 1 mmol/l zinc acetate. Ahigh viscosity gel was formed soon after mixing. 80 μl were injected ineach pig.

EXAMPLE 3

[0040] The zinc containing gel composition of this invention designatedC was: 20 mg/ml GLP-1(7-37), 1 mmol Zn⁺⁺, 16 mg/ml glycerol, and 3 mg/mlm-cresol. The molar ratio between Zn⁺⁺and GLP-1(7-37) was 0.17.

[0041] This composition was made by mixing 1 ml of GLP-1(7-37) solution(40 mg/ml), adjusted to a pH value of 7.4, with 1 ml of a solutioncontaining 6 g/l m-cresol, 32 g/l glycerol and 2 mmol/l zinc acetate. Ahigh viscosity gel was formed soon after mixing. 80 μl were injected ineach pig.

EXAMPLE 4

[0042] The zinc containing gel composition of this invention designatedD was: 20 mg/ml GLP-1(7-37), 2 mmol/l Zn⁺⁺, 16 mg/ml glycerol, and 3mg/ml m-cresol. The molar ratio between Zn⁺⁺and GLP-1(7-37) was 0.33.

[0043] This composition was made by mixing 1 ml of GLP-1(7-37) solution(40 mg/ml), adjusted to a pH value of 8.7, with a 1 ml of a solutioncontaining 6 mg/ml m-cresol, 32 g/l glycerol, and 4 mmol/l zinc acetate.A high viscosity gel was formed soon after mixing. 80 μl were injectedin each pig.

EXAMPLE 5

[0044] As a non-gel, non-protracted solution of GLP-1(7-37) for use as areference in the absorption studies, the following low concentrated zincfree GLP-1(7-37) composition designated REF was chosen: 1 mg/mlGLP-1(7-37), 16 mg/ml glycerol, 3 mg/ml phenol (pH value: 7.3).

[0045] The results of the absorption studies from Examples 1-5 are shownin the Table below. TABLE % Residual radioactivity Time after Prepara-injection Prepara- Prepara- Prepara- Prepara- tion Hours tion A tion Btion C tion D REF 0 100 100 100 100 100 1 — — — — 42.6 1.5 70.6 — — — —2 — 64.1 76.6 94.7 — 3 36.8 — — — 4.5 4 — 44.4 67.0 91.3 — 5 10.3 — — —1.9 6 — 32.7 60.8 — — 6.5 — — — — 1.7 7 3.4 — — — — 15.5 — — — 59.2 —21.5 — — — 40.3 — 24 — 2.4 12.8 — 1.1 40 — — — 9.1 — T-50% 2.3 3.3 8.419.3 0.8 (hours)*

[0046] As appears from these data, the compositions of this inventionare considerably more protracted than the reference solution.

1. A composition containing a GLP-1 compound which composition is a gel.2. A composition, according to claim 1 , which composition hasthixotropic properties.
 3. Composition, according to claim 1 or 2 ,containing not less than about 2 mg/ml, preferably not less than about 5mg/ml, more preferred not less than about 10 mg/ml of a GLP-1 compoundand, preferably, containing not more than about 100 mg/ml of a GLP-1compound.
 4. Composition, according to any one of the preceding claims,containing a phenolic or an alcoholic aromatic compound.
 5. Composition,according to the preceding claim, wherein the phenolic or alcoholicaromatic compound is a pharmaceutically acceptable antimicrobialpreservative.
 6. Composition, according to the preceding claim, whereinthe pharmaceutically acceptable antimicrobial preservative is benzylalcohol, a cresol, e.g., m-cresol, a phenol, e.g., phenol or resorcinol,or a paraben, e.g., methyl paraben or propyl paraben.
 7. Composition,according to any one of the preceding claims, wherein the thixotropicproperty only or mainly results from the presence of a GLP-1 compound.8. Composition, according to anyone of the preceding claims, wherein thethixotropic property only or mainly results from the presence of a GLP-1compound together with a pharmaceutically acceptable antimicrobialpreservative.
 9. Composition, according to anyone of the precedingclaims, containing divalent metal ions, e.g. zinc, calcium, magnesium orcobalt ions.
 10. Composition, according to the preceding claim, whereinthe metal ions are zinc ions.
 11. Composition, according to anyone ofthe preceding claims, containing 1 zinc ion per molecule of the GLP-1compound or less and, preferably, they contain less than 0.4 zinc ionper molecule of the GLP-1 compound, more preferred they contain between0.4 and 0.1 zinc ion per molecule of the GLP-1 compound and mostpreferred between 0.2 and above 0.1 zinc ion per molecule of the GLP-1compound.
 12. A method for the treatment of diabetes mellitus in amammal in need of such treatment comprising the administration of acomposition according to any one of the preceding claims containing aneffective amount of the GLP-1 compound.
 13. A method, according to thepreceding claim, wherein the administration is performed by subcutaneousinjection.
 14. Any novel feature or combination of features describedherein.